Enhancement of Tomato Plant Growth and Productivity in OrganicFarming by Agri-Nanotechnology Using Nanobubble OxygationYuncheng Wu,∇,†,‡,§,∥ Tao Lyu,∇,§,∥ Bin Yue,§,∥,⊥ Elisa Tonoli,# Elisabetta A. M. Verderio,#,¶ Yan Ma,*,†

and Gang Pan*,§,∥

†Institute of Agricultural Resources and Environment, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China‡Nanjing Institute of Environmental Sciences, China Ministry of Environmental Protection, Nanjing 210000, China§School of Animal, Rural, and Environmental Sciences, Nottingham Trent University, Brackenhurst Campus, NottinghamshireNG25 0QF, United Kingdom∥Centre of Integrated Water-Energy-Food Studies (iWEF), Nottingham Trent University, Nottinghamshire NG25 0QF, UnitedKingdom⊥College of Geography and Environmental Engineering, Lanzhou City University, Lanzhou, Gansu 730070, China#School of Science and Technology, Nottingham Trent University, Clifton Campus, Nottingham NG11 8NS, United Kingdom¶BiGeA, University of Bologna, 40126 Bologna, Italy

ABSTRACT: The development of technology to improve the mineralization of organic fertilizer and to enhance cropproduction is essential to achieve the transition from traditional farming to eco-friendly organic farming. Nanobubble oxygation(NB) was employed for comparison with traditional pump-aerated oxygation (AW) and a control group through both soilincubation and soil column experiments. Plant-available N and P contents in the NB treatment group were higher than those inthe AW and control groups. Enzymatic activities including β-1,4-N-acetyl-glucosaminidase, phosphatase, α-1,4-glucosidase, β-1,4-xylosidase, peroxidase, and phenol oxidase were significantly higher in both oxygation groups compared with the control.The soil microbial biomass, activity, and diversity were also significantly improved due to the oxygation treatment. Additionally,the microbial metabolic functions were shifted in both oxygation treatments compared with the control group. The final tomatoyield increase from the NB treatment group was 23%, and that from the AW treatment was 17%, compared with the control.

KEYWORDS: agricultural sustainability, crop intensification, organic farming, precision farming, oxygen nanobubble

1. INTRODUCTION

Currently, an estimated 124 million people in 51 countries arefacing crises from food insecurity and shortage based on the2018 report by United Nation’s World Food Programme(WFP). One of the greatest challenges is how to increase 50% offood production to ensure that the growing global populationpredicted to be around 10 billion by 2050has enough food tomeet their nutritional needs. Many techniques and approacheshave been developed in order to improve crop growth and yield,a simple approach being to increase the application of chemicalfertilizer in traditional farms.1 However, increased fertilizerusage on farmland can cause groundwater pollution,2 surfacewater eutrophication,3 and nutrient loss4 though runoff orleaching. To avoid the adverse impact on both environment andecosystem,5 it is essential to develop an eco-friendly approachfor the enhancement of agricultural crop production.Organic farming is an ideal environmentally friendly

agricultural system, which relies on organic fertilizers derivedfrom livestock manure, crop residues, or human excreta.6

Organic farming also strives for sustainability by promotingnatural pest control and minimizing environmental pollutionfrom synthetic pesticides and antibiotics.7 However, in organicfarming, the applied nutrients from the organic fertilizer can onlybe utilized by crops after decomposition and mineralization oforganic matter and release of plant-available nutrients, such as

nitrogen and phosphorus. It has been reported that only 35%,39%, and 53% of the plant-available nitrogen can be releasedfrom cow, pig, and chicken manures on farmland over 6 months,respectively.8 As a result, crop production in organic farming hasbeen demonstrated to be up to 25% lower than that inconventional agriculture using chemical fertilizer.9 This slowrelease of mineral nutrients from organic fertilizer has becomethe major yield-limiting factor,10 which indicates that furtherresearch could focus on the acceleration of the mineralization oforganic fertilizer in organic farming.The mineralization is driven by microbial biodegradation

processes, where oxygen is crucial in order to improve thebiodecomposition rate. The soil oxygen content in traditionalfarmland originates mainly from air diffusion, which is alwayslimited, especially in the deep soil layer. Thus, an appropriatemethod to deliver sufficient oxygen into the soil is crucial toimprove microbial activity. The application of aerated water tothe farmland through a drip irrigation system has been used todeliver oxygen to the crop root zone.11 Previous studiesdemonstrated that these approaches could not only enhance

Received: July 2, 2019Revised: August 18, 2019Accepted: September 5, 2019Published: September 5, 2019

Article

pubs.acs.org/JAFCCite This: J. Agric. Food Chem. 2019, 67, 10823−10831

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crop yields but also improve the nutrition quality of fruit.12 Toimprove the soil oxygenation efficiency, the aeration pump wasupgraded from common air pumps and fine bubble diffusers toventuri injectors.13 The main aim of the development of thistechnique was to deliver smaller-sized air bubbles into irrigationwater and to improve oxygen dissolution efficiency. Recently,nanobubble technology (NBs; defined as bubbles withdiameters less than 1000 nm)14,15 has attracted increasingattention due to characteristics of high gas solubility and longlifetime of oxygen in the liquid.3,16 A mixture of micro- andnano-bubbles has been used for oxygation in drip irrigationsystems for water saving and for increasing vegetable yields.17

Air-, oxygen-, and nitrogen-saturated nanobubble waters, usedfor irrigation, have been demonstrated to improve the yield ofsuch plants as lettuce, seed germination, and biomassgrowth.18,19 However, the effect of the nanobubble technologyon the mineralization of organic fertilizer still needs to bedemonstrated.Previous studies have mainly focused on the effects of

oxygation on plant physiology, crop yield, quality, and water useefficiency. Soil oxygation can directly improve the plant rootgrowth and nutrient uptake by providing required oxygen forroot respiration and energy generation.20 However, evaluatingthe effect of oxygation on soil properties is also important inorder to reveal the mechanisms for crop yield enhancement. Ithas been proven that soil microbial structure, activity, andmetabolic functions in the soil could be altered, associated withthe change of soil oxygen content.21 Moreover, enzyme activityin soil is important as it directly influences biochemicalprocessing of soil nutrients.22 Therefore, studying the metabolicfunctioning of the microbial community and soil enzymeactivity, coupled with themineralization of organic fertilizer afterthe oxygation treatment, can help us better understand themechanisms of altered crop growth.To evaluate the effect of the proposed nanobubble oxygation

method on organic fertilizer mineralization and crop growth,tomato plant and cow manure compost were selected as themodel crop and target organic fertilizer, respectively. First, a soilincubation experiment was conducted to (1) investigate theeffect on organic fertilizer mineralization by monitoring theplant available nitrogen (NH4

+, NO3−) and phosphorus

(PO43−); (2) evaluate the influence on soil enzymes activities

related to C-, N-, and P-cycling; and (3) detect the response ofthe metabolic functioning of the soil microbial community.Second, a soil column experiment was set up to (4) study thehypothesized positive effect of nanobubble oxygation on tomatogrowth and yield. From the results, this study aimed todemonstrate a promising agri-nanotechnology, nanobubbleoxygation, for the improvement of crop yields in organicfarming.

2. MATERIALS AND METHODS2.1. Aerated Water and Nanobubble Solution Preparation.

The oxygen nanobubble-aerated water was generated by a nanobubblegenerator (KTM, Nikuni Co., Ltd., Kanagawa, Japan). Briefly, thegenerator was operated by recirculation of a fixed volume of 20 L ofdeionized water at a flow rate of 1000 L/h. The superficial liquidvelocity in the column was 0.035 m/s, and the residence time in thesystem was approximately 2.1 min. Pure oxygen (>99%) and air (v/v =1:1) were injected into the system under the gas flow of 0.45 L/m. Thesystem was run for 5 min before use, and the dissolved oxygen (DO) ofthe irrigation water was approximately 15 mg/L measured by a DOmeter (HQ40d, HACH, USA). In order to set a comparable irrigationwater as the traditional oxygation treatment, pure oxygen and air (v/v =

1:1) were used to aerate 20 L of deionized water under the gas flow of0.45 L/m. The aeration was stopped after approximately 5 min whentheDOof aerated water reached 15mg/L under direct measurement bya DO meter (HQ40d, HACH, USA). Thus, the gas volume used fornanobubble solution and aerated water solution were both around 2.25L.

2.2. Soil Incubation Experiment. A laboratory-scale soilincubation experiment (Figure 1a) was performed in order to

investigate the combined effects of oxygation and organic fertilizerapplication in the soil. Two treatment groups were designed as (1) cowmanure applied soil, irrigated by nanobubble-aerated deionized water(NB), and (2) cowmanure applied soil, irrigated by traditional aerationdeionized water (AW). A control group was set as (3) cow manureapplied soil irrigated by original deionized water (Control). Before theincubation experiment, the cowmanure compost was mixed and passedthrough a 2 mm sieve. The sieved cowmanure compost was mixed with600 g of the topsoil at a rate of 1.5% and then was placed in 1 Ltransparent plastic jars. The soil was compacted to give a bulk density of1.3 g cm−3. The jars were covered with loose lids to allow air circulationbut also to minimize water evaporation. There were 12 replicates jars ineach treatment group, and the incubation lasted for 28 days in anilluminated incubator with a constantly dark environment at 25 °C.During the incubation period, all soil jars were maintained at 65% fieldwater-holding capacity. The weight loss of each jar was checked every 2days, and corresponding irrigation water was added to maintain aconstant soil moisture content.

2.3. Soil Column Experiment for Tomato Growth. To evaluatethe effect of combined oxygation and organic fertilizer application ontomato biomass growth and yield, a greenhouse soil column experimentwas performed (Figure 1b). The experiment was conducted from July1st to September 8th in 2018 in the greenhouse at Brackenhurstcampus, Nottingham Trent University, U.K. The three experimentalgroups were designed as follows: (1) the control group (Control,original deionized water + cowmanure compost), (2) the aerated wateroxygation treatment group (AW, normal bubble-aerated water + cowmanure compost), and (3) the nanobubble oxygation treatment group(NB, oxygen nanobubble-aerated water + cow manure compost). Ineach group, 12 replicated, planted, soil columns were prepared. Eachsoil column was 25 cm high with a diameter of 15 cm. Topsoil (0−20cm, 29% sand, 42% silt, and 29% clay), collected from Embleys Farm inthe U.K., was air-dried and sieved by 2 mm mesh. Then, 5 kg of topsoilwas mixed with 75 g of cow manure compost before the columns werefilled. The same size of tomato seedlings at the 3 to 4 leaf stage werethen transplanted into each pot. The plants were watered every dayduring the experiment to maintain 65% field water-holding capacity.

2.4. Sampling and Analysis. 2.4.1. Nanobubble Analysis. Thesizes (<1000 nm) and distributions of nanoscale bubbles in the

Figure 1. Schematics and photos of the lab-scale soil incubation (a) andsoil column (b) experiments.

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traditional-aerated and nanobubble-aerated deionized waters weredetermined by nanoparticle tracking analysis by ZetaView PMX 120(Particle Metrix, Meerbusch, Germany) software and its correspondingsoftware ZetaView 8.04.02. The samples were collected after 5 min ofpreparation, and the analyses were carried out at room temperature.Each sample was analyzed with a flow cell sensitivity of 70% across twocycles of 11 positions/cycle.2.4.2. Sampling Strategies. For the soil cultivation experiments, soil

samples were collected at day 4, 12, 17, and 28 during the experiment.The soil in three replicated jars from each treatment group werecollected after homogenization by mixing with a glass rod. After siftingthe soil samples through a 2 mm sieve, the soil was air-dried prior to the

determination of plant-available nutrients, N and P. At day 28, each soilsample was divided to three parts for nutrient analysis (part I) and thedetermination of dissolved organic carbon (DOC) and microbialbiomass carbon (part II). The remainder of the soil samples (part III)were used to analyze the soil enzyme activity and microbial communitymetabolic functions. For the soil column experiment, the diameter ofthe stem and the height of tomato plants were recorded at 15, 30, and 45days. Tomato fruit from each treatment was harvested and weighed atday 70.

2.4.3. Soil Chemical Properties. The plant-available nitrogen(NH4

+-N, and NO3−-N) in soil samples was extracted by 2 M KCl

solution according to the method described by Tu et al.23 Plant-

Figure 2. Nanoscale bubbles size and distribution in traditional-aerated (a) and nanobubble-aerated (b) solutions, measured by NTA.

Figure 3. Effect of oxygation on plant-available nutrients, i.e., (a) nitrogen and (b) phosphorus, (c) dissolved organic carbon (DOC), and (d)microbial biomass carbon (MBC), in soil incubation experiments. Control, irrigation with original water; AW, irrigation with traditional pump-aeratedwater; NB, irrigation with nanobubble-aerated water. Different letters above the bars in each figure indicate significant difference (P < 0.05) betweenthree groups in the same sampling day.

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available phosphorus (PO43−-P) was extracted with 0.5 M NaHCO3

following a previously reportedmethod.24 The concentrations of NH4+-

N, NO3−-N, and PO3−-P in the extracts were determined by analysis on

an AQ400 nutrients autoanalyzer (Seal Analytical, Southampton,U.K.). The chloroform fumigation−extraction method was used todetermine themicrobial biomass carbon (MBC). The dissolved organiccarbon (DOC) content of the extract was measured by a ShimadzuTOC-V total organic carbon analyzer (Shimadzu Corp., Kyoto, Japan).2.4.4. Soil Enzyme Activities. In the soil cultivation experiment, the

hydrolytic enzymes, peroxidase, phenol oxidase, α-1,4-glucosidase, andβ-1,4-xylosidase were selected as indicators for C acquisition.25 Theterminal reaction in chitin degradation can be catalyzed by β-1,4-N-acetyl-glucosaminidase, and thus it was evaluated as one of the N-targeting hydrolytic enzymes.26 Phosphatase is the enzyme responsiblefor releasing labile inorganic P for microbes and plants.27 The activitiesof all extracellular enzymes, except for phenol oxidase and peroxidase,were measured by using the MUB-linked model substrate methoddescribed by Zhao et al.28 The phenol oxidase and peroxidase activitieswere measured spectrophotometrically by using L-3,4-dihydroxy-phenylalanine as the substrate in a clear 96-well microplate.2.4.5. Microbial Metabolic Functions. In the soil cultivation

experiment, community-level physiological profiling (CLPP) of thesoil samples was assessed by using Biolog EcoPlate (Biolog Inc.,California, USA).29 A 1000-fold serial dilution of the rhizosphere soilsuspension was made, and 150 μL aliquots were added to each well inthe microplates. Soil particles were not removed, nor allowed to settle,during any step in the extraction or inoculation. The plates were thenpacked into polyethylene bags to reduce evaporation and wereincubated in the dark at 25 °C. Absorbance at 590 nm was measuredon an automated microplate reader (Tecan Group, Ltd., Austria) after24, 48, 72, 96, 120, 144, and 168 incubation hours. Each wellabsorbance value was corrected by subtraction of the optical density of acontrol well. The CLPP data was analyzed, based on the previousstudies,30,31 to calculate the average well color development (AWCD)and Shannon diversity indexes.2.5. Statistical Analyses. The data were assessed with one-way

ANOVA. Duncan’s multiple-range test was applied when one-wayANOVA revealed significant differences (p < 0.05). All data were testedfor a normal distribution and variance homogeneity using Levene’s test.The statistical analyses were performed with SPSS version 13.0statistical software (SPSS, Chicago, IL, USA). In addition, a principalcomponents analysis (PCA) was performed on the correlationmatrix ofCLPP results using Origin Pro 2016 software (OriginLab Corp.,Massachusetts, USA). For PCA analysis, data were standardized by an

autoscalingmethod prior to analysis to ensure that each variable had thesame influence in the analysis.

3. RESULTS

3.1. Nanobubbles Distribution in OxygenatedWaters.The aerated waters prepared for irrigation were analyzed in thenanoparticle-tracking analysis instrument to detect the size anddistribution of the nanobubbles (Figure 2). The concentrationof nanobubbles (<1000 nm) was 4.1 × 107 particles/mL in theaerated irrigation water prepared by the traditional pump(Figure 2a); however, a one-magnitude higher nanobubbleconcentration (7.5 × 108 particles/mL) was observed in thenanobubble-aerated water after 5 min operation (Figure 2b),with 87% below 200 nm in diameter. It should be noted that thedeionized water before any aeration treatment containedundetectable concentrations of nanobubbles (<104 particles/mL; data is not shown).

3.2. Plant-Available Nutrients and Organics. The totalplant-available N (NH4

+ + NO3−) content generally increased

from around 38 mg/kg to 50, 56, and 66 mg/kg at day 28, in thecontrol, AW treatment, and NB treatment groups, respectively(Figure 3a). Compared with the control group, enhancements of12% and 32% total available N were observed in the AW andNBtreatment groups, respectively. It can be noted that theconcentrations of NH4

+ in all three groups remained at a similarlevel (around 11 mg/kg) throughout the experiment. Thesignificantly higher NO3

− content in the soil is the maincontribution for the enhancement in total N content. A similartendency in plant-available P in the soil was detected in allgroups throughout the experiment, where the soil from the NBtreatment group contained significantly higher P concentrations(5.9 mg/kg), followed by AW treatment (4.9 mg/kg) and thecontrol groups (4.4 mg/kg) (Figure 3b). No significantdifferences in the concentrations of dissolved organic carbon(DOC, Figure 3c; range of 67.0−79.5 g/kg) were found in thethree groups at the end (day 28) of the incubation. The amountof microbial biomass carbon (MBC) was significantly affectedby oxygation treatment (Figure 3d). In the AW and NBtreatment groups, MBC concentrations were significantly

Figure 4. Effect of oxygation on soil enzyme activities: (a) β-1,4-N-acetyl-glucosaminidase, (b) phosphatase, (c) α-1,4-glucosidase, (d) β-1,4-xylosidase, (e) peroxidase, and (f) phenol oxidase. Control, irrigation with original water; AW, irrigation with pump-aerated water; NB, irrigation withnanobubble-aerated water. Different letters above the bars in each figure indicate significant difference between the values (P < 0.05).

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increased by 17% and 26%, respectively, compared to thecontrol treatment.3.3. Soil Enzyme Activities. After the soil incubation

experiment, soil enzyme activities were analyzed to understandthe mechanisms of nutrient mineralization. All six enzymesexhibited higher activities in the soil samples from the oxygation(AW or NB) treatment groups (Figure 4). The activities of N-mineralization-related enzyme, β-1,4-N-acetyl glucosaminidase(Figure 4a), and P-mineralization-related enzyme, phosphatase(Figure 4b), were significantly higher in the oxygenated groupsthan in the control. There was no significant difference betweenthe NB and AW irrigation groups in the enzyme activity, thoughfor both the enzyme activity was higher than that for the controlgroup. For the C-cycling-related enzymes, the oxygationtreatments slightly improved the activities of α-1,4-glucosidase(Figure 4c), β-1,4-xylosidase (Figure 4d), and phenol oxidase(Figure 4f) compared with the control groups. Both AW andNBtreatment significantly improved the peroxidase activitycompared with the control samples; however, there was nosignificant difference between them (Figure 4e).3.4. Response of Microbial Metabolic Functions. The

community-level physiological profiling was assessed to evaluatethe response of the microbial metabolic functions to the NBirrigation. The soil microbial diversity and activity were reflectedin the Shannon diversity index and the AWCD values,respectively. Both of these were significantly higher in theoxygation treatment groups compared to the control group(Table 1). The levels of microbial diversity and activity weresimilar between NB and AW treatment groups.

The biochemical properties of the 31 carbon sources in themicroplates were organized into six groups (guilds), namely,miscellaneous, carbohydrates, polymers, carboxylic acids, aminoacids, and amines/amides, in order to reduce the complexity ofthe data obtained. Overall, the capabilities of the soil microbialcommunities for carbon utilization were strengthened in theoxygation treatment groups (Figure 5a). However, only aminoacids showed significantly higher utilization in AW and NBtreatment groups than in the control group. Further evaluationwas used to transform the multivariate vectors into twouncorrelated principal component vectors (Figure 5b). Thetwo-dimensional PCA of the community-level of physiologicalprofiles explained 68.6% of the total variance, with the firstprinciple component having a greater power of separation(42.7%). The data from the oxygation treatment groups locatedin the upper right section of the figure were significantly differentfrom the control group, shown on the left of the plot. Theanalysis of the loading of carbon sources on PC1 showed thatAW and NB treatments were indeed factors that influenced the

catabolic diversity of microbial communities. The data betweenNB and AW groups are generally overlain (Figure 5b).

3.5. Tomato Growth and Yield. The soil oxygationtreatments from both AW and NB significantly improved thetomato growth as measured by improved stem diameter (Figure6a) and plant height (Figure 6b) at the early stage of plantgrowth on day 15 and 30. However, by day 45, these differenceswere not significant in both AW and NB treatment groupscompared with the control group (Figure 6a and b). The tomatobiomass from the NB oxygation group yielded a significantlyhigher value (around 547 g/plant) than that (around 447 g/plant) from the control group, an enhancement of some 22%(Figure 6 c and d). The tomato yield from AW oxygenationtreatment group was around 523 g/plant, which was 17% higherthan the control group.

4. DISCUSSIONIn the process of agricultural production, fertilization andirrigation are key practices for improving the yield of crops.32 Toreduce environmental issues caused by overuse of chemicalfertilizers without influencing crop yield, organic fertilizer hasbeen recommended as a partial, or even complete, substitute.33

Oxygation (aerated irrigation) is an irrigation technology whichis well recognized to enhance crop yield by improving theaerobic environment of the root zone and to increase rootuptake of water and nutrients.34 However, studies focused onthe effect of both soil oxygation and organic fertilizer applicationon crop growth are still limited. In the present study, twooxygation methods, irrigation by traditional pump-aerated waterand nanobubble-aerated water, were applied in growth oftomatoes with organic fertilizer.The majority of the nutrients, stored in organic form in

organic fertilizers, cannot be directly utilized by the crops. Therelease of plant-available nutrients, such as N and P, fromorganic matter involves biological decomposition processes,which are highly dependent on the oxygen content and moisturelevel in the soil. Oxygation offers soil sufficient water and oxygenat the same time, and thus the plant-available N and P contentcan be increased, such as occurs under ventilation treatment.35 Itsupports the present finding that the irrigation of both normalpump-aerated and nanobubble-aerated waters significantlyincreased the release of plant-available N and P from organicfertilizer (Figure 3). Specifically, nitrogen content organicfertilizer can release NH4

+ through the biodegradation processunder the aerobic condition. If the oxygation approachsubstantially supplies the oxygen, NH4

+ can be transformed toplant-available NO3

− through the nitrification process.36 Thus,the substantially higher content of NO3

− compared with NH4+

(Figure 3a) may be due to the dominant nitrification processunder such an aerobic environment. Moreover, the significantlyhigher nutrients under NB oxygation may be attributable to thelarge amounts of nanoscale bubbles in NB-aerated irrigationwater (Figure 2). The NB has a low buoyancy and long lifetime,where the filled air or oxygen can be slowly dissolved into the soilinterstitial water and sustainably supply the oxygen37 requiredfor the mineralization of organic fertilizer. The effective oxygensupply by NBs may also result in the high speed of the organicfertilizer mineralization, and plant-available N in the soilachieved the highest value in day 17 (Figure 3a). The similarlevel shown in day 28 may be caused by the thoroughly plant-available N release under the oxygation treatment. It isdifferentiated from the normal pump-aerated water, where theoversaturated oxygen can escape from the irrigation water

Table 1. Microbial Metabolic Functional Diversity andAverage Well Color Development (AWCD) Level from theBiolog EcoPlate Analysis in Control, Pump-Aerated Water(AW) and Nanobubble-Aerated Water (NB) IrrigationGroupsa

group Shannon diversity (H′) AWCD (590 nm)

Control 3.344 ± 0.003 b 2.0 ± 0.1 bAW 3.387 ± 0.001 a 2.4 ± 0.1 aNB 3.388 ± 0.005 a 2.5 ± 0.1 a

aDifferent superscript letters beside the number indicate a significantdifference at P < 0.05.

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quickly to the atmosphere, resulting in a comparatively reducedoxygenation effect and speed on the rhizosphere environment.Agriculture practices, such as irrigation, can influence soil

microenvironment and result in the shift of soil micro-

organisms.38 Higher soil aeration was reported to stimulatemicrobial biomass and change community composition inpaddy fields,39 findings which support the determination ofimproved microbial biomass, activity, and diversity in the

Figure 5. Microbial community carbon source utilization levels (a) and the principal component analysis (PCA) ordination of the carbon sourceutilization patterns (b) from Biolog EcoPlates incubated for 168 h. Control, irrigation with original water; AW, irrigation with pump-aerated water;NB, irrigation with nanobubble-aerated water. Different letters above the bars in each figure indicate a significant difference between the values (P <0.05).

Figure 6.Tomato plant growth, i.e., (a) stem dimension and (b) plant height, and (c, d) tomato biomass yield at the end of the soil column experiment.Control, irrigation with original water; AW, irrigation with pump-aerated water; NB, irrigation with nanobubble-aerated water. Different letters abovethe bars in each figure indicate a significant difference between the values (P < 0.05).

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aerated irrigation groups in this study (Figure 3d and Table 1).The differences of microbial metabolic functions in the soilsamples were indicated by the utilization of 31 kinds of carbonsources during the Biolog microplate analysis.40,41 The clearlydifferentiated metabolic function groups between the aeratedirrigation group and the control (Figure 5) further demonstratedthat the oxygenation treatments not only boosted microbialactivity but also played a constructive role in increasingfunctional diversity of soil microbial communities.42 Eventhough the microbial metabolic functions (Figure 5b) wereundifferentiated between the normal pump-aerated and nano-bubble-aerated irrigation treatments, gene level differences inthe soil microbial communities may be significant, which need tobe further studied.Soil extracellular enzymes aremainly synthesized and secreted

by soil microorganisms.43 Changes in metabolic function anddiversity of soil microbial community might cause thefluctuation of soil enzyme activities. Previous studies foundsome soil enzyme activities were greater in soils treated byaeration than in those without.35,44 In this study, we found soilenzyme activities were increased by oxygation treatment. Themechanism may be due to the stimulation of microbial growthand the increase in the activity of the extracellular enzyme−organo complex.45 Among the six enzymes that we measured(Figure 4), the activities of C-cycling enzymes (α-1,4-glucosidase, β-1,4-xylosidase, phenol oxidase, and peroxidase),a N-cycling enzyme (β-1,4-N-acetyl-glucosaminidase), and a P-cycling enzyme (phosphatase) suggest a shift toward increasedC acquisition as N and P becomes readily available for plantgrowth. Increases in enzyme activities may reflect and stimulatesoil microbial activity, thereby increasing the quantities ofnutrients available to plants.46 However, the similar enzymesactivities were observed in the two oxygation treatments, whichmay due to the relative short soil incubation time before thesampling.Plant height and stem diameter were significantly increased in

the early stage of tomato plant growth (15, 30 days) in bothoxygation treatments (Figure 6). The result is consistent withprevious studies showing that oxygation treatment can improvethe rate of organic fertilizer mineralization and can result in a fastcrop growth.47 However, the plant growth (stem diameter andheight) achieved the same level at the final stage after the fruithad ripened (Figure 6). Similar results were also found for thetomato cultivation under aerated irrigation,12 which may due tothe same amount of fertilizer application in all groups. It hasbeen reported that the tomato yield with oxygation treatmentwas around 19% higher when compared to non-oxygationtreatment.48 In the present study, the AW treatment withtraditional pump-aerated irrigation reached a similar increase(17%) of tomato production, while the nanobubble-aeratedirrigation achieved around a 23% improvement in yield (Figure6 c), which is comparable to the losses (up to 25%) generallyattributed to the transition from traditional farming usingchemical fertilizer to organic farming using organic fertilizer.9

Therefore, the present study provides a promising eco-friendlyagri-nanotechnology, with which to increase crop production inorganic farming. Nevertheless, further studies should beconducted to evaluate the effect of NB oxygation on organicfertilizer mineralization and crop growth directly in the soilcolumn experiment before the application. Notably, in thepresent study, the plant-available nutrients and microbialcommunities from the nanobubble irrigation treatment areonly slightly different to those obtained by conventional pump-

aerated irrigation group. The relatively larger tomato yield mayalso be due to the synergistic functions of improvement inorganic fertilizer mineralization and plant physiology modifica-tion by the nanobubbles.18,19 Thus, the plant gene alteration andfruit nutrition changes will need to be further studied.In conclusion, the nanobubble oxygation treatment for

organic farming was evaluated for the improvement of organicfertilizer mineralization and tomato production, compared withthe traditional pump-aerated oxygation technique and withunoxygenated control groups. Levels of plant-available N and Pwere substantially improved, associated with the stimulation ofsoil enzymatic activity due to the oxygation treatment.Moreover, this treatment, in an organic farming context,significantly enhanced the soil microbial biomass, activity,diversity, and metabolic functionality. Even through thedifferences between the nanobubble oxygation and traditionpump-aerated oxygation treatments were not always significant,final tomato yields improved by approximately 23%, while thepump-aerated oxygenation treatment gave an improvement incrop yield of 17%, when compared to the control group. Theresults indicated that the proposed agri-nanotechnology,nanobubble oxygation, is a potentially promising approach tostimulate mineralization of organic fertilizer and thus improvecrop growth, during a transition from using chemical fertilizer toorganic fertilizer for organic farming.

■ AUTHOR INFORMATION

Corresponding Authors*E-mail: myjaas@sina.com (Y.M.).*E-mail: gang.pan@ntu.ac.uk (G.P.).

ORCIDGang Pan: 0000-0003-0920-3018Author Contributions∇These authors are co-first authors and contributed equally tothis work.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSThis work was funded by the National Natural ScienceFoundation of China (41701339), the China PostdoctoralScience Foundation (2017M621672), the Medical Technolo-gies and Advanced Materials Strategic Theme and theSustainable Future Research Theme at Nottingham TrentUniversity. We thank Mick Cooper for proof reading.

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